Methods and compositions for purification or isolation of microvesicles and exosomes

ABSTRACT

The invention relates to the isolation or extraction of exosomes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/471,191, filed on Jun. 19, 2019, which is a National Phase entry ofInternational Application No. PCT/AU2017/051460, filed Dec. 22, 2017,which claims priority to Australian Patent Application No. AU2016905369, filed Dec. 23, 2016, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to cellular microvesicles, particularly exosomes,to molecular modelling and to chromatographic methods for separation andisolation of molecule including ion exchange chromatography.

BACKGROUND OF THE INVENTION

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction.

Microvesicles are a heterogeneous collection of biological structuresbound by membranes. These structures have a lipid bilayer and they mayreside within a cell or in an extracellular environment. Microvesiclesrange in size from about 20 nm to 1000 nm.

Exosomes are small lipid bilayer vesicles, secreted by multiple celltypes, and ranging in diameter from 20 to ˜150 nm (as determined byelectron microscopy (EM)). Unlike other cell-derived microvesicles,exosomes are formed within the intracellular space and then secreted bythe cell (Raposo and Stoorvogel, 2013).

The lipid composition of exosomes is distinct from that of the cell oforigin, but is still somewhat characteristic of the cell type from whichthey originate. In addition, exosomes show some common lipid featuresindependently of their origin (Urbanelli et al., 2013).

Depending on their origin, exosomes may contain a wide variety of“cargo”, including but not limited to peptides, proteins, lipids,transcription regulators, messenger RNA (mRNA), noncoding RNA (ncRNA)and double stranded DNA (dsDNA).

Exosomes are now considered as a means by which an originating cell(i.e. the cell from which the exosome oiiginates) can communicate with atarget cell, in situations such as neuronal synapses, immune regulation,angiogenesis, tissue regeneration and epigenetic modulation. Suchcommunication can be localized or remote. Indeed, exosomes from mother'smilk can modulate immune and other functions in the new-born. Suchcommunication can even be cross-species, for example, but not limitedto, bovine milk exosomes modulating immune and other functions inhumans.

Exosomes can be sourced from various biofluids, including saliva, urine,blood, blood plasma, (breast) milk, synovial fluid ascites fluid, sap,and fruit extracts.

In summary of the above, it appears that the unique characteristics ofexosomes is driven by their size, cell uptake by endocytosis, their“cargo”, and that they deliver “messages” that become biologicallyactive in the target cell.

There is mounting evidence that exosomes may play a role in the onsetand perpetuation of various diseases. Exosomes collected from biologicalsamples can provide diagnostic information on the current state of adisease. The concentration of RNA in exosomes can be up to 60 timesgreater than that extracted directly from body fluids such as blood,driving the potential for their utility in diagnostics. For example,identification of oncogenic RNA signature within exosomes isolated fromhuman fluids could potentially yield a diagnosis of an abnormal statewell before invasive cancer develops.

Recently it has been shown that the function of therapeutic mesenchymaltype cells is not dependent upon the cell itself, but rather paracrinefactors produced by the cell, including exosomes, establishing thatexosomes are one part of the circulating cell secreted factors that canmediate the effect seen in some cell therapies in a cell-free manner(Rani et al., 2015).

Therapeutically useful exosomes collected from the conditioned media ofcells undergoing in vitro culture can also be used as a cell-freetherapeutic, but there is a need for robust, flexible method ofisolation from their source fluids (Chen et al., 2010).

In addition, exosomes could provide a natural delivery vehicle packedwith therapeutic proteins and RNA when cell-based expression systems aretailored specifically.

Exosomes are invisible under normal optical microscope. Technologiesused for detection of these ultramicroscopic particles like exosomes andmicrovesicles include electron microscopy (EM), flow cytometry, dynamiclight scattering (DLS), and nanoparticle tracking analysis (NTA). It hasbeen reported that, depending on the method used, the reported absolutenumber of extracellular vesicles in a liter of blood can vary by as muchas five orders of magnitude (van der Pol et al., 2010).

While exosomes are abundant and expected to be useful either in adiagnostic or therapeutic sense, the means to isolate and purifyexosomes has limited their clinical application to date.

In general, existing methods for exosome isolation are characterized bytwo groups:

-   (i) affinity capture due to some (possibly unique) selective    external feature of the vesicle (e.g. glycoprotein marker,    transmembrane proteins like tetraspanins (CD9, CD63, CD81, and CD82)    and MHC class I and II, and cytosolic proteins like heat shock    proteins (HSP-70 and HSP-90))-   (ii) isolation or purification based on some biophysical property of    the exosome (e.g. size, density, zeta potential, highly curved    surface that contains lipid-packing defects, membrane curvature    sensors)

The limitations of these techniques are set forth in the table below

Method Application Limitations Ultra- Differential centrifugationScalability, operational centrifugation Density gradient centrifuga-complexity, EV aggregation (UC) tion (Greening et al., 2015) PolymerPolyethylene glycol based Co-precipitation of protein assistedprecipitation (Rider et al., contaminants, need for precipitation 2016)removal of polymers after precipitation Immunoaffinity CD9, CD63, CD81and Isolation of sub-population capture EpCam antibody based ofexosomes, lack of single capture (Greening et al., standard maker for2015) (U.S. patent applica- exosome, issues with tion 20150010913)removal of antibody bound to exosome Tangential flow Sequentialfiltration Requirement of additional filtration (Heinemann et al., 2014)steps to further purify exosomes.

US2013/0273544 (Vlassov) and US201510192571 (Ghosh) both disclose theuse of polymers including poly ethylene glycol and certainpolysaccharides as volume excluding polymers for the precipitation ofmicrovesicles from a solution. US20160216253 discloses the use ofheparin for isolation of extracellular vesicles.

There is a need for an improved method for isolation of exosomes, inparticular a method that provides for an improved yield of exosomes, andthat is scalable for pharmaceutical grade manufacture or production ofexosomes, and that avoids other limitations of the prior art discussedabove.

SUMMARY OF THE INVENTION

The invention seeks to address one or more of the above mentioned needsor limitations and in one embodiment provides a method for obtaining anisolate or composition of exosomes. The method includes the followingsteps:

-   -   providing a liquid that includes exosomes;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic or            electron-rich group; and        -   wherein the array of exosome-binding ligands enables            exosomes to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of exosomes to the exosome-binding ligands;    -   separating the substrate from the liquid;    -   thereby obtaining an isolate or composition of exosomes.

In another embodiment, there is provided a method for obtaining anisolate or composition of exosomes. The method includes the followingsteps:

-   -   providing a liquid that includes exosomes;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic or            electron-rich group at pH of 4-8, preferably pH 5 to 8,            selected from the group consisting of CO₂H. CH₂OH, CH₂OSO₃H,            B(OH)₂, and CH₂OP(O)(OH)₂, or those groups in ionized form            as dictated by their relevant pKa;        -   wherein each ligand is spaced apart from other ligands by            about 4-5 Å;        -   wherein the array of exosome-binding ligands enables            exosomes to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of exosomes to the exosome-binding ligands;    -   separating the substrate from the liquid;    -   thereby obtaining an isolate or composition of exosomes.

In another embodiment there is provided a method for obtaining anisolate or composition of microvesicles. The method includes thefollowing steps:

-   -   providing a liquid that includes microvesicles;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic or            electron-rich group; and        -   wherein the array of exosome-binding ligands enables            microvesicles to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of microvesicles to the exosome-binding ligands;    -   separating the substrate from the liquid;    -   thereby obtaining an isolate or composition of microvesicles.

In another embodiment, there is provided a method for obtaining anisolate or composition of microvesicles. The method includes thefollowing steps:

-   -   providing a liquid that includes microvesicles;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic or            electron-rich group at pH of 4-8, preferably pH 5 to 8,            selected from the group consisting of CO₂H, CH₂OH, CH₂OSO₃H,            B(OH)₂, and CH₂OP(O)(OH)₂, or those groups in ionized form            as dictated by their relevant pKa.        -   wherein each ligand is spaced apart from other ligands by            about 4-5 Å        -   wherein the array of exosome-binding ligands enables            microvesicles to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of microvesicles to the exosome-binding ligands;    -   separating the substrate from the liquid;    -   thereby obtaining an isolate or composition of microvesicles.

The invention also provides an apparatus or device including a substrateas described above that enables execution of the above described method.

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are intended toprovide further explanation of the invention as claimed. Theaccompanying drawings are included to provide a further understanding ofthe invention and are incorporated in and constitute part of thisspecification, illustrate several embodiments of the invention, andtogether with the description serve to explain the principles of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates a projected end on view of a predicted structure ofcellulose/cellufine sulfate;

FIG. 1B illustrates a side on view of a predicted structure ofcellulose/cellufine sulfate;

FIG. 1C illustrates a projected end on view of a predicted structure ofcellulose/cellufine phosphate;

FIG. 1D illustrates a side on view of a predicted structure ofcellulose/cellufine phosphate;

FIG. 2A illustrates a projected end on view of a predicted structure ofchitosan (A);

FIG. 2B illustrates a side on view of a predicted structure of chitosan(A);

FIG. 3 illustrates a predicted structure of poly (methyl vinylether-maleic anhydride);

FIG. 4 illustrates a predicted structure of heparin;

FIG. 5 illustrates a predicted structure for 3-APBA;

FIG. 6 illustrates a hyaluronic acid predicted structure;

FIG. 7 illustrates a chondroitin sulfate predicted structure;

FIG. 8 illustrates a polyvinyl sulfate predicted structure;

FIG. 9 illustrates a poly glutamic acid predicted structure;

FIG. 10 illustrates a poly aspartic acid predicted structure;

FIG. 11 illustrates a polyserine predicted structure;

FIG. 11(A) illustrates a biotin-C3-poly-4-hydroxybutyl-β-asparaginepredicted structure;

FIG. 11(B) illustrates a biotin-C3-poly-4-hydroxybenzyl-β-asparaginepredicted structure;

FIG. 12 illustrates a poly D L Serine sulfate predicted structure; and

FIG. 13 illustrates a general structural model.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention is concerned with the design of reagents and methods forimprovements in the isolation of extracellular vesicles, especiallyexosomes, from biological sources including conditioned cell media, invivo fluids, tissues and biopsies, and extracts or fractions or isolatesthereof. In particular, the invention is concerned with reagents andmethods that provide for improved capture of extracellular vesicles froma source and improved release, resulting in reproducibly higher yieldsof extracellular vesicles. As described herein, the inventors havedetermined heretofore unknown characteristics required in substances forobtaining improved yields of extracellular vesicles, in particular,exosomes, from biological sources. This work enables the selection andor design of substances for use in methods of isolating microvesiclesand exosomes.

“Extracellular vesicles (EVs) or micro vesicles (MVs)” generally referto a heterogeneous in vivo collection of membrane bound biologicalstructures having a diameter from about 20 to 1000 nm.

“Exosome” generally refers to a vesicle having a lipid bilayer and adiameter of about 20 to 200 nm as measured by EM.

“Binding efficiency” generally refers to a measure of the proportion ofexosomes bound by a substrate. It is the difference of the amount ofexosomes remaining in a sample after application of the method of theinvention and the amount of exosomes in a sample before application ofthe method of the invention.

“Elution efficiency” generally refers to a measure of the proportion ofexosomes eluted from a substrate. It is the difference of the amount ofexosomes in the eluate after elution according to the method of theinvention and the theoretical amount of exosomes bound to the substrate.

“Yield” generally refers to the number of exosomes released from theeluate as a proportion of the total number of exosomes before theapplication of the method of the invention.

“Comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

In a first embodiment the invention provides a method for obtaining acomposition or an isolate of exosomes. The method includes the followingsteps:

-   -   providing a liquid that includes exosomes;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic or electron            rich group; and        -   wherein the array of exosome-binding ligands enables            exosomes to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of exosomes to the exosome-binding ligands;    -   separating the substrate from the liquid;    -   thereby obtaining a composition or an isolate of exosomes.

In another embodiment there is provided a method for obtaining anisolate or composition of microvesicles. The method includes thefollowing steps:

-   -   providing a liquid that includes microvesicles;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic or            electron-rich group; and        -   wherein the array of exosome-binding ligands enables            microvesicles to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of microvesicles to the exosome-binding ligands;    -   separating the substrate from the liquid;    -   thereby obtaining an isolate or composition of microvesicles.

According to this embodiment, a substrate is provided having a surface.An array of exosome-binding ligands is provided on the surface so as toenable contact of the exosome-binding ligands with the outer surface ormembrane of a microvesicle when the liquid is contacted with thesubstrate.

The array of exosome-binding ligands is more or less an orderedarrangement of exosome-binding ligands on the substrate surface.Typically the array takes the form of a plurality of exosome-bindingligands that are positioned across at least a part or region of thesubstrate surface.

An array may be in the form of a planar array or a linear array.

A planar array generally extends in 2 dimensions so as to define anarea. A planar array may generally include a region of exosome-bindingligands having a density of about 1 to 10 ligands per nm², preferablyabout 5 ligands per nm². This spacing can be determined for example byatomic force microscopy. Other regions of the array may have a higher orlower density of exosome-binding ligands. Thus in one embodiment thereis provided a method for obtaining a composition or an isolate ofexosomes or microvesicles. The method includes the following steps:

-   -   providing a liquid that includes exosomes;    -   providing a substrate having a surface, the surface having an        planar array of exosome-binding ligands;        -   wherein each ligand is in the form of an anionic or electron            rich group; and        -   wherein the planar array of exosome-binding ligands enables            exosomes to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of exosomes to the exosome-binding ligands;    -   separating the substrate from the liquid;

thereby obtaining a composition or an isolate of exosomes,

wherein the planar array preferably includes a region of exosome-bindingligands having a density of about 1 to 10 ligands per nm², morepreferably about 5 ligands per nm.

A linear array generally extends in one dimension. In one embodiment, alinear array may be formed where exosome-binding ligands are provided ona polymer that has a generally linear arrangement on a substratesurface. In another embodiment, in use, the polymer may extend from thesubstrate into a solvent. In a linear array, the exosome-binding ligandsare generally provided in a range of from 1 to 5 ligands per nm,preferably about 2 ligands per nm. This spacing can be determined forexample by atomic force microscopy or mass spectrometry. Thus in anotherembodiment there is provided a method for obtaining a composition or anisolate of exosomes or microvesicles. The method includes the followingsteps:

-   -   providing a liquid that includes exosomes;    -   providing a substrate having a surface, the surface having a        linear array of exosome-binding ligands;        -   wherein each ligand is in the form of an anionic or electron            rich group; and        -   wherein the linear array of exosome-binding ligands enables            exosomes to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of exosomes to the exosome-binding ligands;    -   separating the substrate from the liquid;

thereby obtaining a composition or an isolate of exosomes,

wherein the exosome-binding ligands are preferably provided in a rangeof from 1 to 5 ligands per nm, more preferably about 2 ligands per nm.

In further embodiments, it has been found that a substrate that has anarray of exosome-binding ligands distributed across the substratesurface that interfaces with a microvesicle outer membrane surface, inwhich an exosome-binding ligand is spaced no more than about 3.5 to 10angstroms apart from another exosome-binding ligand, forms a highlyefficient exosome-binding surface. As explained further below, where thearray is provided to the substrate surface by one or more polymers thatinclude the exosome-binding ligands, the measurement of 3.5 to 10angstroms, preferably greater than 2 angstroms, particularly about 4 to6 angstroms, with reference to the spacing of exosome-binding ligands,refers to the spacing observed in the lowest energy state of the polymeras determined by the methods exemplified herein.

As described further herein, the invention provides a structural modelof an arrangement of exosome-binding ligands that predicts substratesthat are highly efficient exosome binders. According to the model,highly efficient exosome binders are generally substrates that haveanionic or electron rich groups spaced apart by about 4 to 6 Angstromsas measured according to the methodology on which the structural modelis based. The invention enables one to predict the binding efficiency ofa substrate for an exosome or microvesicle through the identification ofanionic or electron rich groups and by modelling the structure of thesubstrate utilizing the methodology for the derivation of the structuralmodel herein.

Preferably an exosome-binding ligand is spaced more than 2 angstromsapart from another exosome-binding ligand. More preferably anexosome-binding ligand is spaced 3.5 to 6, or 4 to 6 angstroms, morepreferably 3.5, 4, 5 or 6 angstroms apart from another exosome-bindingligand in the array.

Typically the majority of ligands, preferably 60%, or 70% or 80%, morepreferably 90%, still more preferably 95%, 96%, 97%, 98% or 99% ofligands are spaced more than 2 angstroms, and no more than about 10angstroms apart, preferably 3.5 to 6, or 4 to 6 angstroms from anotherligand in the array.

According to the invention, the arrangement of the exosome-bindingligands in the array may have various levels of order. In oneembodiment, there may be a very high order of arrangement ofexosome-binding ligands within the array. For example, each ligand maybe spaced apart from other ligands in the array by a precise distance of4 angstroms. Further, the position of each ligand, and/or the spacesbetween ligands may define a particular pattern. In such a higher orderof arrangement, generally all of the ligands are evenly spaced apart inthe array. As described herein, it has been found that a surface havingan array of exosome-binding ligands with a higher order of arrangementgenerally has a reproducibly higher exosome or microvesicle bindingefficiency.

In other embodiments, there may be a lower order of arrangement ofexosome-binding ligands within the array. For example, the majority, butnot all of the ligands may be spaced apart from each other by more than2 angstroms, and no more than about 10 angstroms apart, preferably 3.5to 6, or 4 to 6 angstroms from another ligand in the array. Further, ofthis majority of ligands, there may be variability as between thespacing of these ligands in the array. For example, some ligands may bespaced apart from others by 3.5 angstroms, others by 4 angstroms, othersby 5 angstroms, others by 6 angstroms. In such an arrangement, theposition of each ligand, and/or the spaces between the ligands may notdefine a discernible pattern. However, it has been found that such alower order array has useful binding efficiency for binding exosomes.

As would be appreciated, the extent of order amongst exosome-bindingligands can be thought of as a local-order and meta-order. By this wemean that looking at the array of exosome-binding ligands at a distancethere might seem to be a randomness to the location of the ligands (i.e.they are not in clear orthogonal arrays) (meta-order) yet the meandistance between ligands might be relatively tightly distributed arounda mean value (local-order). In this invention it is discovered that ahigher-order of local-order is preferred whereas the extent ofmeta-order is of less importance. Therefore, in certain embodiments, thearray may include regions of meta-order, provided that the arraycontains at least one region of local-order.

An array may be formed on the substrate surface by a variety of methods.A higher order array may be formed by deposition of exosome-bindingligands at precise positions on the substrate surface. Examples includecrystalline silicon, ceramic or polymer surfaces using lithography,plasma immersion ion implantation and deposition (PIII&D), atomic forcemicroscope, laser, electrical etching, low-density plasma reactive ionetching, wet etching, electron microscope or other means known toproduce a functionalized surface containing exosome-binding ligands.

In another embodiment, an array may be printed on a substrate surface.Examples include functionalizing the substrate surface with a chemicalpad and then further derivatizing such pads with exosome-binding ligandmoieties which are now chemically bound to such pads.

In another embodiment, the array is provided to the substrate surface byone or more polymers that include the exosome-binding ligands. In thisembodiment, the substrate may be integrally formed from the polymerthereby forming a substrate surface. Additionally, or alternatively, thepolymer may be coupled or otherwise bound to the substrate, therebyforming a substrate surface, for example via biotin/streptavidinbinding.

In embodiments where the substrate is formed from the polymer, thesubstrate surface may be treated so as to activate that polymer forformation of exosome-binding ligands on the polymer.

In other embodiments, a polymer including exosome-binding ligands iscoated onto the substrate, thereby forming exosome-binding ligands onthe substrate surface.

As stated above, and described in detail further herein, where theexosome-binding ligands are to be provided in an array by one or morepolymers, those polymers are selected according to whether, at thelowest energy, the ligands they present are observed in silico to bespaced apart from each other by more than 2 angstroms, and no more thanabout 10 angstroms apart, preferably 3.5 to 6 angstroms, or 4 to 6angstroms apart. Briefly, the in silico observation is derived fromsubjecting a sub structural unit (typically hexamer to dodecamer, butmay be larger) of a polymer, complete with anionic or electron-richgroups to geometry-minimization calculations (MM+algorithm) so as todisplay a most stable (lowest energy) three-dimensional structure, andtherefore the structure likely to be involved in exosome binding. Thedistance between ligands can then be measured in silico to determine thelikelihood of binding to exosomes. When the energy-minimized structureof the modeled oligomer bears a suitable number of ligand pairs whereinthe separation of electron-rich or anionic ligand pairs is of a distancefrom 2 to 10 angstroms, then the polymer that this modeled oligomerrepresents will be suitable for binding exosomes or microvesiclesaccording to this invention.

Thus in another embodiment there is provided a method for obtaining acomposition or an isolate of exosomes or microvesicles. The methodincludes the following steps:

-   -   providing a liquid that includes exosomes or microvesicles;    -   providing a substrate having a surface, the surface having an        array of exosome    -   binding ligands;        -   wherein each ligand is in the form of an anionic group or            electron rich group; and        -   wherein the array of exosome-binding ligands enables            exosomes to bind to the substrate;    -   contacting the liquid with the substrate in conditions enabling        the binding of exosomes or microvesicles to the exosome-binding        ligands;    -   separating the substrate from the liquid;

thereby obtaining a composition or an isolate of exosomes ormicrovesicles,

wherein the exosome-binding ligands are observed in silico to be spacedapart from each other by more than 2 angstroms, and no more than about10 angstroms apart, preferably 3.5 to 6 angstroms, preferably 4 to 6angstroms apart in an energy minimization model described herein.

The order of an array formed from exosome-binding ligands provided onone or more polymers is generally dependent on the heterogeneity of thepolymers with respect to the order of monomers within each polymer andthe length of each polymer. Preferably, the polymers for use in theinvention have little heterogeneity, or otherwise, more or less,homogeneity with respect to the monomer order and polymer length.Preferably the polymers are linear.

In this regard, synthetic polymers, rather than polymers derived fromnatural or biological sources are preferred. This is because polymersfrom biological sources, such as certain polysaccharides, havesignificant heterogeneity with respect to either polymer length,branching, monomer order and even monomer functionalization. Asdescribed herein, this heterogeneity generally contributes to a lowerefficiency of binding of exosomes or microvesicles and a lower exosomeor microvesicle yield. Therefore, in one embodiment the polymer is notan unfractionated or heterogeneous natural or biological source,particularly of heparin.

More preferably, with respect to monomer content, the polymers have auniform order of monomers, particularly where the polymer comprisesdifferent monomer species, and wherein only some of the monomer speciescontain exosome-binding ligands. It is particularly important that theuniformity of monomer order creates a uniformity in the spacing of theexosome-binding ligands that are presented to the outer membrane surfaceof the exosome or microvesicle. This generally requires that there is auniform stereochemistry of exosome-binding ligands for interaction withthe exosomes or microvesicles.

It is preferred that at least 25% of monomers forming a polymer,preferably 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% of monomersforming a polymer have an exosome-binding ligand that is arranged on thesubstrate surface to enable binding to an exosome or microvesicle outermembrane.

It is particularly preferred that all of the monomers forming a polymerpresent exosome-binding ligands that are spaced about 3.5 to 6angstroms, 4 to 6 angstroms, preferably, 3.5, 4, 5 or 6 angstroms fromeach other to the outer membrane surface of an exosome or microvesicle.

In certain embodiments where a polymer consists of more than onemonomeric species, a species may provide one type of exosome-bindingligand and another monomeric species may provide another type ofexosome-binding ligand.

Further, depending on the length of a monomer species, a monomer speciesmay provide more than one exosome-binding ligand (which may be the sameor different), provided that the ligands provided on the monomerspecies, in their presentation to the exosome or microvesicle outersurface are spaced about 3.5 to 6 angstroms, or 4 to 6 angstroms,preferably, 3.5, 4, 5 or 6 angstroms from each other.

A surprising finding of the invention is that exosomes andmicro-vesicles can be separated from other components of an in vivo orin vitro formed biological fluid on the basis of charge, and inparticular, a positive charge on the exosome or microvesicle. This issurprising because it had been generally understood that most cellmembranes and lipid bilayers have a net negative charge, particularlyarising from lipid polar head groups, membrane sugars and proteins thatare generally negatively charged at physiological pH.

The exosome-binding ligand utilized in the above described embodimentsis in the form of, or consists of an anion or an electron rich group.Exosomes are generally stable at physiological pH of 7.4+/−1-1.5.Therefore, the exosome-binding ligand is generally an anion, orotherwise an electron rich group at a pH of about 4 to 8 or 5 to 8.

The exosome-binding ligand may be an inorganic or organic anion orelectron rich group. Examples of inorganic anions include anions thatcontain a sulfur, selenium, boron, nitrogen or phosphorus atom andinclude sulfates, selenates, phosphates, phosphonates, phosphinates.

Preferably the ligand is an organic anion or electron rich group.Examples include sulfated alcohols and amines, amides, sulfonamides,carboxylate groups, alcohols, ethers, anhydrides, selenates, imides,acylsulfonamides, phosphonates, phosphinates, phosphates, etc.

In one embodiment, an array of exosome-binding ligands according to theinvention may be a combination of organic and inorganic anions orelectron rich groups.

An important finding of the invention as described herein is that wherea polymer is utilized to provide the array of exosome-binding ligands,the polymer is selected on the basis of its capacity to present ordisplay a uniformly spaced plurality of exosome-binding ligands to amicrovesicle outer membrane surface, rather than on the basis of someother characteristic of the polymer backbone. In this regard theinvention is distinguished from other approaches, described previously,which have utilized polymers to precipitate microvesicles from solution.Those approaches have typically selected polymers on the basis ofaqueous solubility and volume exclusion characteristics. Exemplarypolymers utilized in these approaches are polyethylene glycol[US2013/0273554 and US2013/0337440] and polysaccharides [US20150192571].

According to the invention, the polymer may be a polysaccharide,polyethylene terephthalate or peptide with side chains suitable forderivatization with anionic or electron-rich groups, such aspolyaspartic acid, polyserine, polythreonine, polyasparagine, nucleicacid or other organic molecule, such as polyethers. (PEGS), sulfatedpolyvinyl alcohols, polyphenols, polyphenylboronic acids, or otherpolyaromatics or polyheteroaromatics, preferably synthetic.

In embodiments where the polymer is derived from a natural or biologicalsource, and the polymer is known to have significant source-dependentheterogeneity, specifically with respect to polymer length, monomercontent and order, it is particularly preferred to fractionate thenatural or biological source so as to decrease heterogeneity andincrease homogeneity for a polymer of desired length, monomer content ororder.

Typically the polymer has a molecular weight of 10 kDa to 900 kDa kDa,preferably 20 to 50 kDa for certain polysaccharides such as heparin, or100 to 200 kDa for certain other molelcules, an example being chitosan.

Particularly preferred polymers are synthetic or homogeneous natural orbiological sources of polysaccharides selected from the group ofpyranoses, wherein monomers include (but are not limited to) dextrose,glucose, galactose, glucosamine, galactosamine, mannose, ribose,arabinose, xylose, lyxose, and amino sugars of the like.

Polymers may be provided in the form of peptides. A particularlypreferred peptide is polyaspartic acid. As described herein, polyglutamic acid may be less preferred given that the spacing of theanionic side chains exceeds the preferred range of the invention of 4 to6 angstroms. Other preferred peptides include polyserine, polythreonine,polyasparagine, polycysteic acic, polyselenocysteic acid, andD-configured amino acids of the same.

The polymer backbone may be aqueous soluble or insoluble.

In a particularly preferred embodiment the polymer contains a singlemonomer species having a length approximating the length of a glucosemonomer (about 1.5 nm), or is a glucose monomer or derivative thereofhaving a length of about 1.5 nm, and wherein each monomer includes ananionic or electron rich group arranged so that each group is spacedapart by about 4 to 5 angstroms, and wherein the polymer contains about100 to 400 monomers, preferably about 250 to 300 monomers, and/or has amolecular weight of about 40 to 60 kDa, preferably about 45 to 55 kDa.

In a preferred embodiment the polymer is a linear chain cellulosemolecule wherein each monomer for presentation to an exosome outermembrane surface is functionalized with an anionic group or electronrich group, preferably a sulfate group.

It is not necessary that the entirety of the substrate surface should becovered by or contain the array of the exosome-binding ligands. In oneembodiment, the substrate consists of an exosome or microvesicle bindingregion having an array of exosome-binding ligands on the region toenable exosomes or microvesicles to bind to the binding region. Othersurfaces of a substrate may be engineered so as to preclude binding ofexosomes. For example, these “non binding” regions may be constructed sothat, in use, they do not present exosome-binding ligands to exosomes ormicrovesicles. Such an arrangement of exosome binding and non bindingregions may be useful in the formation of devices for production,detection or monitoring of exosomes. Such devices may includemicrofluidic devices, or larger devices for cell culture or medicaldevices for clinical use.

The substrate may take the form of a range of shapes or configurations.

The surface of the substrate may be flat, curved, indented or porous.The surface may be formed within a device, tube or well, or on a bead.

In one embodiment, the substrate surface is brought into communicationwith one or more sensors for sensing the presence of, properties of, orsize of exosomes or microvesicles bound to such substrate, usingsuitable nanotechnology analytical techniques such as atomic forcemicroscopy, transmission electron microscopy (TEM), Auger emissionspectrometry (AES) laser mass spectrometry, X-ray photon spectroscopy.

In another embodiment the substrate surface is in communication withmeasurement means to determine useful parameters of theexosome-substrate (or micro-vesicle-substrate) combination such as oneor more of (i) presence or absence of exosome (and thereby number anddensity of exosomes) (ii) size of exosome (iii) conductivity of exosome(iv) a surface marker of exosome (v) rigidity of exosome (vi) charge ofexosome or (vii) some other useful parameter through such means as oneor more of (a) electrical probes, (b) imbedded laser (c) electric forcemicroscopy (d) magnetic force microscopy (e) scanning gate microscopy orother suitable techniques. The substrate may form the measurement means.

The method of the invention utilizes substrates and exosome-bindingligands defined above to isolate microvesicles, in particular exosomes,from a biological source on the basis of charge. As described above, itis a surprising finding of the inventors that exosomes andmicro-vesicles can be separated from other components of a biologicalfluid on the basis of charge, and in particular, on the basis of apositive charge of the exosome or microvesicle. On the basis of thisfinding, the inventors have utilized an ion exchange chromatographyapproach to the isolation of exosomes and microvesicles.

According to the invention the liquid containing the exosomes ormicrovesicles is contacted with the substrate in conditions enabling thebinding of exosomes or microvesicles in the liquid to theexosome-binding ligands. This then binds exosomes or microvesicles tothe substrate, which is the basis for the subsequent separation ofexosomes or microvesicles from the liquid, as resulting from theseparation of the substrate from the liquid.

The conditions for enabling binding of exosomes or microvesicles to theexosome-binding ligands generally require a consideration of saltcontent and pH of the liquid that contains the exosomes ormicrovesicles. Generally the pH of the liquid is defined by the pHstability of the exosomes or microvesicles, and this pH stability rangeis about 4 to 8 In some embodiments, the liquid containing the exosomesor microvesicles may be pre-processed prior to contact with thesubstrate. Such processing may include for example a pH adjustment tooptimize the binding interaction with the exosome-binding ligands. Forexample, it may be necessary to adjust the pH of the solution tophysiological pH level of 7.2 to 7.4. This ensures that the moieties onthe exosomes or microvesicles that are bound by the exosome-bindingligands are cationic and able to bind to the anions or otherwiseelectron rich groups of the exosome-binding ligands.

Processing may also include salt adjustment, potentially saltminimization of the liquid, for example by a dialysis step, beforecontacting the liquid with the substrate.

Typically the liquid containing the exosomes or microvesicles will havea pH in the range of 7.2 to 7.4 and a salt content of 0.1 to 0.5M,preferably 0.15M and phosphate content of 0.01M prior to contact withthe substrate.

Further processing may be required to remove cellular debris orcontamination that may interfere with the binding of the exosomes ormicrovesicles to the exosome-binding ligands. This processing mayinvolve centrifugation at 500 g, with or without filtration to about0.22 micron.

In one embodiment, the liquid may be in the form of a biological fluidwhich is centrifuged to form a supernatant and a microvesicle-containingpellet and the supernatant is discarded and the pellet is re-suspendedin an ion exchange binding buffer for contact with the substrate. Such abuffer generally has a pH in the range of 7.2 to 7.4 and a salt contentin the range of 0.1M to 0.2M.

Filtration may also be employed to deplete microvesicles of a particularsize from the liquid prior to contact of the liquid with the substrate.For example, the liquid may be fractionated to remove exosomes with adiameter greater than 100 nm prior to contact of the liquid with thesubstrate. Alternatively, this step could be performed at the completionof the elution of microvesicles from exosome-binding ligands (asdescribed further below).

The liquid and substrate are brought into contact for a period of timesufficient to enable exosomes to bind to the exosome binding ligands.Generally this is from 1 minute to 16 hours and can be in around 15minutes. It could be less than 1 minute for example less than 30seconds.

The contact of the liquid with the substrate may be established in avariety of formats. For example the liquid may be percolated through asolid resin or bead, captured, and the percolation and capture cyclerepeated for a pre-determined number of cycles.

As discussed above, a particular advantage of the invention lies in therevelation that there is disparity among charged polymers, (particularlythose derived from natural sources that have an inherent heterogeneity)vis a vis their capacity to bind to exosomes or microvesicles, and thatit is possible to obtain significant improvements in binding efficiencyby judicious selection of substrates or polymers according to theinvention herein. In particular in the work described herein, theinventors have shown that polymers that perhaps might otherwise havebeen considered equally efficient for exosome or microvesiclebinding—because in common they are all negatively charged atphysiological pH—have varying levels of efficiency and that the variouslevels of binding efficiency can be explained by the arrangement of thenegative charges presented to the outer membrane surface of an exosomeor microvesicle.

In particular, as described herein, the inventors have determined thatthe overall isolation yield, which arises principally from differencesin theoretical binding efficiency, ranges from about 4.3% (as observedfor hyaluronic acid) to up to 98% (as observed for cellufine sulfate).Further, by determining a structural and binding model for the polymerstested for binding efficiency, the differences in binding efficiencyhave been explained in terms of the arrangement of anion and electronrich groups presented to the exosome outer membrane surface, enablingselection of optimal substrates or polymers for exosome binding, and inparticular those more likely to provide a higher binding efficiency.

By implementing this work in an exosome or microvesicle isolationprocedure, the invention enables a broader range of binding options thancould otherwise be applied for obtaining an acceptable yield of exosomesor microvesicle. This is particularly useful where there are limits asto the pH or salt conditions that can be applied in a binding buffer.

Further the invention enables a more efficient binding procedure,potentially enabling shorter binding periods, or where multiple bindingcycles would otherwise have been required, fewer binding cycles. Theseimprovements enable the ion exchange approach to exosome or microvesicleisolation to be credibly translatable to high throughput processing, amanufacture practice that will ultimately be required to enablepharmaceutical scale production of exosomes.

A final step in the binding phase of the method is, as foreshadowedabove, to separate the substrate from the liquid, thereby separatingexosomes (which are bound to the exosome-binding ligands on thesubstrate) from the liquid. This separation may be achieved actively byremoving the substrate from the liquid (for example by centrifuging asubstrate in the form of beads), or passively by passing the liquid overthe substrate, (thereby depleting exosomes from the liquid as the liquidseparates from the substrate).

A particularly important advantage of the invention is that it enableselution of exosomes and/or microvesicles from a biological source so asto produce an eluate that consists primarily of exosomes and/ormicrovesicles and elution buffer. This is particularly important forregulating the quality control of the end product. In comparison, otherapproaches to date for isolation of exosomes result in the exosome beingpurified with a precipitating agent, for example, in the form ofpoly-ethylene glycol, or a polysaccharide or fragment thereof, togetherwith an enzyme for degradation of the polysaccharide. These approachesrequire further manipulation of the end product to remove thesecontaminants, and this further manipulation, such as centrifugation orfiltration, may deleteriously affect the exosomes.

It is a surprising finding of the invention that exosomes andmicrovesicles that have bound via higher affinity or higher avidityinteractions to the substrates of the invention may be uniformly elutedfrom exosome-binding ligands, enabling release of the exosomes from theligands. Specifically, as described herein, it has been found thatselected but unrelated substrates, namely cellufine sulfate, chitosanand poly (methyl vinyl ether-maleic anhydride) have an improved exosomebinding efficiency and the exosomes bound thereto may be commonly elutedby utilizing elution buffers having a range of salt concentrations or pHranges within which exosomes remain stable.

Thus in one embodiment, the method includes the further step of elutingexosomes or microvesicles from the exosome-binding ligands to releasethe exosomes or microvesicles from the substrate after the substrate isseparated from the liquid.

According to the invention, the exosomes or microvesicles may be elutedby contact of the substrate with an elution buffer. Typically an elutionbuffer has a salt content from about 0.5 to 4M, or from 0.5 to 2M,depending on the pH of the buffer system. This salt content establishesa preferential binding between the anionic species in the elution bufferand the outer membrane of the exosome or microvesicle resulting inelution.

In further embodiments, the salt content of the elution buffer may bethe same as the binding buffer, and the elution buffer may have a higherpH than the binding buffer. This results in elution of the exosomes ormicrovesicle from the exosome-binding ligands as the pH of the buffersystem approaches the isoelectric point of the moieties on the exosomeor microvesicle which bind to the exosome binding ligands. Thus in oneembodiment an elution buffer has a salt content of about 0.5 to 2M and apH of about 7.2 to 7.4 pH units.

In another embodiment, the elution buffer may include oligomers of thepolymer forming the substrate surface, said oligomers having a higheranionic charge or higher electropotential than the exosome-bindingligands on the polymer thereby resulting in elution of exosomes ormicrovesicles. For example, where the exosome binding ligands areprovided on the substrate surface in the form of a cellulose sulfatepolysaccharide, the elution buffer may include shorter chain celluloseoligomers having a higher amount of sulfation.

In another embodiment the elution buffer may include a competitiveligand that has a higher affinity than the exosome binding ligand on thesubstrate for an exosome or microvesicle. For example, an exosome can beeluted from a boronic acid polymer using an elution buffer containing anexcess of a sugar, preferably fructose, which has a higher affinity thanboronic acid for exosomes, thus eluting exosomes from the substrate.

In one embodiment, the elution buffer is applied to the substrate onceonly. In other embodiments the elution buffer may be continuously cycledfor a pre-determined number of cycles.

In one embodiment, the method may include the further step of separatingthe released exosomes or microvesicles from the substrate. This step isparticularly required where the exosomes or microvesicles remain insolution with the substrate at the completion of elution. One example iswhere the exosomes or microvesicles are eluted from exosome-bindingligands arranged on a substrate in the form of a bead and the bead is tobe removed from the released exosomes or microvesicles. In otherembodiments, the physical separation may result simply from the exosomesbeing eluted from the substrate, as for example may occur in a columnchromatography.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

EXAMPLES Example 1—Exosome Isolation Using Polymer Based Substrates

1.1 Test Material

The test material used were cell culture conditioned medium derived frommurine hypothalamic neuronal cell line GT 1-7 cultured in Opti-MEMcomplete culture media (Opti-MEMS Reduced Serum Medium+10% exosomedepleted fetal calf serum+Penicillin-Streptomycin+GlutaMAX) for 48-72hrs.

1.2 Test Method

Freshly harvested cell culture conditioned medium were centrifuged(500×g for 5 minutes) to remove cells and large debris, then thesupernatant was filtered using 0.22 μm syringe filter to remove largevesicles and apoptotic bodies. The filtered conditioned medium wasquantified for particle concentration using Nanoparticle Trackinganalysis (NTA) on NanoSight NS300.

Table 1 describes a panel of polymer based substrates for which thebinding and elution efficiency and overall isolation yields weredetermined.

Chitosan was 200 kDa (Biotin-) Chitosan (custom made). Viroadem beadswere Ademtech Viro-Adembeads. Superdex® 75 was from Sigma Aldrich#S6657. BA-Magbeads were from Chemicell 1504-5 SIMAG-Boronic Acidmagnetic particles. PEG-Control: Biotin-PEG24 (as control for3-APBA/4-APBA which strictly speaking are Biotin-PEG24-3APBA andBiotin-PEG24-4APBA (custom made). Mag Bead Control was from SpherotechSVM-40-10 Avidin Coated Magnetic Particles. A Norgen Biotek Cell CultureMedia Exosome Purification Kit (#60600) was used. Chitosan, heparin,3-APBA, 4-APBA, PEG control and hyaluronic acid were Biotin-X coupled toSpherotech SVM-40-10 Avidin Coated Magnetic Particles.

The ligand immobilized on substrate at optimized concentration wereincubated with 1 ml quantified test material for 5 hours at 4° C. on atube rotator (100 rpm) to test the ability to affinity capture exosomes.The ligand immobilized-solid substrate was separated post-incubation andthe concentration of the particles were determined in solutionpost-capture (unbound exosomes) using NTA on NanoSight NS300. Theseparated ligand immobilized-solid substrate bound to exosomes wereincubated in elution solution overnight at 4° C. on a tube rotator (100rpm) to elute bound exosomes. The ligand immobilized-solid substrate wasseparated post elution incubation and the concentration of particleswere determined in solution using NTA on NanoSight NS300.

1.3 Data Acquisition

The particle concentration was determined using NTA on NanoSight NS300.The data acquisition strategy employed was to acquire 5×30 second videosin static mode operation. NanoSight software then analyzed the 5independent 30 second video acquisitions to prepare an experimentalreport with the mean (+/−standard error) particle concentration data.

1.4 Test Read Outs

We measured particle concentration at three experimental stages

(i) Pre-affinity separation (i.e. particle concentration in testmaterial before capture)

(ii) Post affinity capture in solution (i.e. particle concentration intest material after capture representing unbound exosomes)

(iii) Post elution (i.e. particle concentration in solution afterelution of ligand captured particles)

1.5 Data Analysis Methodology

The test read out data obtained was used to determine four parameters:

-   -   ligand binding efficiency (i.e. number of particles in the test        material captured by exposure to the ligand immobilized solid        substrate)    -   ligand elution efficiency (i.e. proportion of that ligand        captured particle that were eluted)    -   the overall process yield

1.6. Results

In the optimized binding condition, cellulose sulfate ligand, in theform of cellufine sulfate, showed a theoretical binding efficiency of77.90% with the number of theoretical bound particles being 1.31×10¹⁶(Table 1). The number of particles recovered after elution fromcellufine sulfate beads was 1.64×10¹⁶ with the elution efficiency beingquantified by the instrument at 125% (Table 1). The overall processyield for cellufine sulfate ligand was 98% (Table 1). Note the elutionefficiency of 125% was subject to a variety of quantification datavariance due to operational and other factors. One would expect theactual elution efficiency to trend towards less than 100%.

Viroadembeads showed a theoretical binding efficiency of 66.13% with thenumber of theoretical bound particle being 1.11×10₁₀ (Table 1). In theelution conditions tested, we were able to elute 41% of the particlescaptured by Viroadembeads with a mean size of 232.4 nm (Table 1). Theoverall process yield for Viroadembeads was 27% (Table 1).

Unexpectedly, the overall particle isolation yield forultracentrifugation based exosome isolation was 7.5% (Table 1). Thisyield was drastically lower compared to the overall isolation yieldobserved with ligands reported in this invention. Nordin et al hasreported exosome yield by ultracentrifugation to be 10% (Nordin et al.,2015). Furthermore, 4 L of adipose stem cell derived CM yielded 0.9 mgexosomes by ultracentrifugation, requiring 23 L CM and 276-280 hrsprocessing to generate a single human dose likely to be 5 mg. Incomparison, ligand described in this invention with an exosome yield of80% could generate a single human dose of 5 mg from less than 4 L ofadipose stem cell derived CM with shorter processing time when processedin chromatography mode. This further highlights the superiority of theligands described in this invention to isolate exosomes compared to thecurrent gold standard isolation method of ultracentrifugation.

Example 2—Determining EMIT Structural and Functional Model

To determine the likelihood of optimal exosome binding amongfunctionalized polymers, an in silico energy minimization protocol wasfollowed. An oligomer representing a subsection of the polymer, whichmay range from a hexamer to a dodecamer in size, was drawn using thecorrect stereochemical configuration of ligands, where known, such as inthe case of chitosan or cellufine sulfate, or defined, such as in thecase of polyaspartic acid. Where the configuration was random orunknown, such as in the case of poly (methyl vinyl ether-maleicanhydride), then a random oligomeric structure was used in kind. Thestructures were then subjected to energy minimization calculations (MM+or AMBER molecular mechanics model, HyperChem version 7.5), using aPolak-Ribiere descent function. The number of computational iterationsneeded to determine the energy minimum was typically 1000-2000 but if anenergy minimum was not reached, the calculation was allowed to continueuntil such a minimum was achieved. At this point the arrangement wasviewed from several angles and the positioning of the ligands wasassessed in terms of mutual distance between, for instance, sulphuratoms in the case of sulfated polysaccharides, or anhydride carbonyloxygens, in the case of poly (methyl vinyl ether-maleic anhydride. Thesecondary structure of the polymer in these cases illustrate arrangementof the backbone, which consists of the 6-12 monomer units, into ahelical shape upon which the binding moieties project outwards. Itshould be understood that these energy-minimization calculationsrepresent fractional subsections of the polymers, rather than thepolymers as a whole, due both to software/processing limitations, andalso to any variability in overall polymer size. Further, the MM+/AMBERtype calculations are conducted in the absence of solvating moleculessuch as water or other components likely to be encountered in vivo or ina practical sense. Because these calculations are indeed performed onlyin the sense of the ligand-functionalized oligomers themselves, themethod eliminates inconsistencies and unpredictable components thatwould be present in more complex media, and thus can be consideredinternally consistent. Moreover, the results obtained, with ligandspacing within the minimized structures, display a pattern consistentwith observations of exosome binding efficiency.

Example 3—Predicted Structures of Cellufine Sulfate and CellulinePhosphate

Cellufine sulfate is a synthetically derived cellulose sulfate particle,commercially available as Cellufine Sulfate (JNC Corporation, Japan).The synthesis method of cellufine sulfate preparation is part ofEuropean patent EP0171086 Å2. In brief, non cross linked celluloseparticle were developed from glucose polymer made of 250-300 glucoseunit as raw polymer. Cellulose particle were then sulfated using sulfatereagents to derived cellulose sulfate beads (cellufine sulfate). Thetotal sulfur content on the particles is 900 μg/g dry weight.

The cellufine sulfate heptamer model minimizes as shown (FIGS. 1(A) and1(B)), projected end-on and side-on, with sample atom-atom measurementsbetween neighboring sulfate oxygen species. The sulfate moieties(measured sulfur to neighboring sulfur) are approximately 6.7-7.9 Åapart, with the charged oxygens capable of moving within 4.5 Å (asindicated in the side-on projection). This model suggests that to bindexosomes, there must be both sufficient clearance (outward projectionfrom the helix) and hydrogen bond-forming capability, such as between analcohol moiety and an anionic or neutral, electron-rich oxygen. Theexosome surface molecules to which ligands form hydrogen bonds arelikely therefore to be hydrogen bond donors, such as alcohols, such asglycols within phospholipids or other major components of cellmembranes. In this case a primary alcohol group (CH₂OH) is projectedbetween two anionic groups, such as sulfates, forming a pincer-typehydrogen bond network, wherein the alcohol hydrogen can interact withtwo oxygen atoms at a time. The degenerate nature of the sulfate oxygensmeans that the probability of interaction is greater, (three persulfate, six in total for a pair of sulfates) and thus increases thestrength of the hydrogen bond network for binding. See FIGS. 1(A) and(B).

Cellufine phosphate, similarly, is a synthetically derived cellulosephosphate particle. It is also commercially available as CellufinePhosphate (JNC Corporation, Japan). Represented as a dodecamer forillustrative purposes, FIG. 1(C) and (D) indicate end-on and side-ondepictions of the helical structure in the model. The model indicatesthe partially charged nature of the phosphate groups (CH₂OP(O)(OH)₂) asmono anions, effectively CH₂OP(O)(OH)O⁻ consistent with the pH range ofanticipated binding. In this case as well as for the cellufine sulfate,a primary alcohol group (CH₂OH) is projected between the anionicphosphates to form the pincer-type hydrogen bond network. The alcoholhydrogen can interact with two oxygen atoms at a time. The degeneratenature of the phosphate oxygens is similar to that of the sulfates,though the pH dependence of protonation degree of the phosphate willdictate the probability of interaction. As shown in FIGS. 1(C) and 1(D),two of the oxygens are degenerate and increase the strength of thehydrogen bond network for binding.

Example 4—Predicted Structure of poly (methyl vinyl ether-maleicanhydride)

The anionic polymer, poly (methyl vinyl ether-maleic anhydride)(poly(MVE-MA) is commercially available as Viroadembeads (Ademtech,France). The brief synthesis method for Viroadembeads is described below(Sakudo et al., 2009b). Small (300 nm in diameter) monozide magneticparticles (reducing sedimentation and offering a broad surface) with ahigh ferrite content (allowing for face separation under a magneticfield) prepared by the grafting of poly(MVE-MA) in dimethyl sulfoxide(DMSO)/phosphate buffer 5/95 solution for 3 hours at 37° C.

In the case of poly(methyl vinyl ether-maleic anhydride) (PMVEMA), thestereochemistry of substitution on both the furandione and backbonemethoxy substitutions is random and undefined. For the purpose ofobtaining an energy-minimized structure, a uniform and consistentstereochemical configuration was used, resulting in a sheet structurewherein altemating furandione moieties point away from the backbone.Separation between neighboring carbonyls, which could together act ashydrogen bond acceptors for interaction with phospholipids, isapproximately 4.4 Å. See FIG. 3.

Example 5—Predicted Structure of Chitosan

Chitosan, a natural polysaccharide resulting from the N-de-acetylationof chitin, contains 60-100% N-deacetylated monosaccharide units. Theenergy-minimized structure of chitosan forms a helix with the aminogroups located towards the inner face, placing alcohol moietiesprojected outwards from the helix, as shown below, end-on. A side-onprojection illustrates the position of the neighboring primary alcoholgroups, proposed here as the binding moieties capable of interactingwith the phospholipid of exosomes. These are roughly 4.6-5.0 Å apartarranged around the periphery of the helix. See FIGS. 2A and 2B.

Example 6—Predicted Structure of Heparin

Heparin, which is less sulfated than heparan, consists of glucuronicacid, galactosamine, and iduronic acid moieties. Heparin contains moreiduronic acid than heparan, which carries more glucuronic acidmonosaccharides. Therefore, an energy-minimized model of heparin isarbitrary since prediction of the geometry of binding moieties cannot beexpected to be consistent throughout the polymeric structure.Assumptions are therefore predicated on an arbitrary arrangement ofmonosaccharide units, nevertheless the energy minimized structure alsoforms a helical superstructure, from which sulfate groups are projectedoutwards. Distances between neighboring sulfur atoms are between 5 and 6Å as illustrated. Given that heparin would contain fewer sulfates thanthat depicted, it is possible that this would result in lowered bindingefficiency than, for instance, cellufine sulfate, which contains a moreconsistent sequence of monosaccharide units. See FIG. 4.

Example 7—Predicted Structures for 3-APBA and 4-APBA

The compound 3-APBA refers to biotinylated (PEG-24)-3-aminophenylboronicacid, which, according to Table 1, bound exosomes poorly. Its4-aminophenylboronic acid-based analogue (4-APBA in the above table) wasalso ineffective. Both compounds require sonicating prior to use,suggesting that in media they tend to fold in on themselves and formcolloids or other suspensions that disfavor binding to exosomes. The PEGcontrol (simple polyethylene glycol) would have different physicalcharacteristics in solution and would not associate in similar manner.

Organic polymers bearing equally spaced arrays of anionic groups,including boronic acids (such as poly-(3-acrylamidophenylboronic acid)occupy energy-minimized geometry that allows neighboring aryl groups toπ-stack, with the anionic appendages arranged in fairly close proximity.This example, with random stereochemistry off the core polyacrylamidebackbone, illustrates both features, with neighboring boron atoms lessthan 4 Å apart. See FIG. 5.

Example 8—Hyaluronic Acid Predicted Structure

Hyaluronic acid, which consists of alternating saccharide unitsconnected through the 3- and 4-position, cannot easily form a tighthelical structure wherein the negatively charged carboxylates form asimilar conformation to those anionic groups present, for instance, incellufine sulfate or heparin. The alternating disaccharides minimize toa less ordered, non-helical array, placing the carboxylates inrelatively random positions. This lack of order may contribute to thecompound's inability to bind exosomes efficiently. Here, thecarboxylates are illustrated in green on a space-filling model toillustrate their lack of proximity to each other. See FIG. 6.

Example 9—Chondroitin Sulfate Predicted Structure

Chondroitin sulfate exists in various forms of 100 or more alternatingglucuronic acid and N-acetylgalactosamine units, wherein the alternatingmonosaccharide units are joined at various positions. For this reason itis impossible to create a specific model by which one can predict itsability to bind exosomes. However, an MM2+-minimized model of an octamerillustrates that neighboring sulfate and carboxylate groups projectoutwards from the core backbone may be sufficiently exposed tofacilitate interaction with phospholipids. See FIG. 7.

Example 10—Polyamino Acids Predicted Structure

Polyamino acids, another potential exosome binding ligand type, includepolyglutamic acid, polyaspartic acid, polyserine, and the like.

Gamma-polyglutamic acid is predicted to adopt a sheet form withcarboxylate groups situated perpendicularly to the backbone,approximately 12 Å apart: this distance between potential bindingmoieties may not be sufficient to permit efficient interaction withphospholipids. Therefore this polymer is predicted to be a lowefficiency exosome binder.

Polyaspartic acid can be prepared in all L-form, all D-form, or as aconfigurational mixture by various synthetic methodologies. Forillustrative purposes, an octamer of the poly-L-Asp is depicted asN-acetyl-Asp7NH2. In this case, the energy-minimized molecule appears asa series of alternating loops, upon which the carboxylates projectoutwards, with neighboring CO₂-groups between 4.5 and 5.0 Å apart(carbon to carbon distance). According to the structural modelpolyaspartic acid is predicted to be an efficient exosome binder.

Poly-β-asparagine derivatives can be prepared in all L-form, all D-form,or as a configurational mixture by various synthetic methodologies asdescribed in example 11.

Based on this model, as well as observations of secondary structure inmultiserine regions of naturally occurring proteins, syntheticpolyserine would also be expected to exist in a series of alternatingloops. In this case the proposed exosome binding moities, (electron richalcohol moieties similar to those of chitosan), occupy similarneighboring distances to other exosome-binding polymers.

Sulfation of serines and other hydroxyl group bearing amino acids (e.g.tyrosine, threonine), to form polyserine sulfates, has been described inU.S. Pat. No. 4,444,682A (1984) by Rivier and Penke of the SalkInstitute, by means of acetylsulfuric acid salts. Preparation ofpolyserine sulfate would thus deliver a peptide-like analogue of highlysulfated polysaccharides such as cellufine sulfate. The geometry of thismolecule, by virtue of its asymmetry, adopts a more helical shape thanthat of the unsultfated polypeptide. As depicted in randomized sidechain form (as would be seen in poly-D,L-Ser-sulfate) neighboringsulfate moieties arrange themselves outwards in a manner similar to thatdisplayed by polysaccharides. Intersulfur atom distances are withinsimilar range: 4.5-6.5 Å. See FIGS. 9 to 12.

Example 11(a). Biotin-C3-poly-4-hydroxybutyl-β-asparagine and example11(b) biotin-C3-poly-4-hydroxybenzyl-β-asparagine

Synthesis is accomplished by treating the polymeric anhydride biotinC3-AspAn, (biotin C3-polysuccinimide) with 1-hydroxybutylamine or4-phenoxyethylamine as indicated, resulting in the ring-opened polymericβ-asparagine derivatives. For Illustrative purposes, an octamer ofpoly-β-1-hydroxybutylasparagine, FIG. 11(A), is used to indicate theenergy-minimized conformation of example 11(a) and FIG. 11 (B)illustrates the energy-minimized conformation of example 11(b). Bothexamples bound exosomes, illustrating the range of effective ligandseparation.

Example 12—General Structural Model

A general model for polymer binding to exosomes can thus be representedas such: a helix or sheet with suitably spaced, projecting anionic orhydrogen-bonding capable groups extending outwards, where the backboneconsists of a repeating template, and the anionic groups may berepresented where R═CO₂H, CH₂OH, CH₂OSO₃H, B(OH)₂ and CH2OP(O)(OH)2, orthose groups in ionized form as dictated by their relevant pKa, and thelike to which the exosome is capable of binding. The model explains thatpolymers with high binding efficiency for exosomes bear anionic orelectron rich groups wherein their mutual separation is between 4-5 Å.See FIG. 13.

Example 13—Polyvinyl Sulfate Predicted Structure

The sulfur atoms in polyvinyl sulfate minimize themselves (in nonamermodel) to about 4.2-4.5 angstroms apart. See FIG. 8.

Example 14 Prediction of Binding Efficiency of Exosome-Binding LigandsBased on Structural Model

The general structural model at example 11 was utilised to predict thebinding efficiency of exosome binding ligands, including those tested inthe previous examples. The results are shown in Table 2.

TABLE 1 Overall Isolation EAL Binding EAL elution yield (% recoveredTheoretical efficiency efficiency exosomes = number of (TheoreticalNumber of Theoretical Number of eluted bound % of bound eluted % ofeluted exosomes(F) *100/ exosomes exosomes = exosomes exosome number ofinput Test Ligand (A-C) (A-C)*100/A) (F) (F*100/(A-C)) exosomes (A))Cellulose Sulfate 1 ml 1.31 × 10¹⁰  77.90%  1.64 × 10¹⁰ 125%  98%Chitosan (500 ug) 5 × 107 8.75 × 10⁹  48.60% 8.75 × 10⁹ 100%  48.60%  Viroadembeads 250 ul 1.11 × 10¹⁰  66.13%  4.6 × 10⁹ 41% 27% Heparin (500ug) 5 × 107 3.5 × 10⁹   22% 3.43 × 10⁹ 98% 21.80%   Superdex 200 ulNegative  1.8 × 10⁹ 17.60%   3-APBA (500 ug) 5 × 107 1.5 × 10⁹  7.20% 2.5 × 10⁹ 166%  12% 4-APBA (500 ug) 5 × 107 Negative 3.35 × 10⁹ 12% BA-Magbeads (500 ul) 4.2 × 10⁹ 15.10% 3.33 × 10⁹ 79% 12% PEG-Control (forBA ligands) 7.5 × 10⁹ 36.00%  2.5 × 10⁹ 33.30%   12% Hyaluronic acid(500 ug) 5 × 107  5 × 10⁹ 22.70%  7.8 × 10⁸ 15.60%   4.30%  Mag BeadControl 4.0 × 10⁹ 23.80%  1.4 × 10⁹ 35% 8.30%  Norgen (1 ml CM) Not Not7.92 × 10⁸ Not 7.70%  applicable applicable applicable Norgen (7 ml CM)Not Not 2.38 × 10⁹ Not 2.20%  applicable applicable applicableUltracentrifugation(1 ml CM) Not Not 1.36 × 10⁹ Not 7.55%  applicableapplicable applicable

TABLE 2 Comparison of ligands LEAP tested and ligands predicted by EMITstructural model LEAP Binding Test EMIT Ligand Backbone Group outcome*prediction Cellulose Polysaccharide Sulfate Positive Binding SulfateCellulose Polysaccharide Phosphate Not tested Binding phosphate ChitosanPolysaccharide —OH Positive Binding Heparin Polysaccharide SulfatePositive Less effective Hyaluronic Polysaccharide Carboxylate NegativeLess acid effective Chondroitin Polysaccharide Sulfate and Not testedBinding sulfate Carboxylate Dextran Polysaccharide —OH Not tested Lesseffective Dextran sulfate Polysaccharide Sulfate Not tested Bindingpoly(methyl Polymer Carbonyls Positive Binding vinyl syntheticether-maleic anhydride) (PEG)₂₄ Polymer Boronic acid Negative No BindingBoronic Acid synthetic Boronic Acid Polymer Boronic acid Not testedBinding Polymer synthetic Polyaspartic Peptide Carboxylate Not testedBinding acid Substituted Peptide —OH Positive Binding poly-β- asparaginePolyserine Peptide —OH Not tested Binding Polyserine Peptide Sulfate Nottested Binding sulfate Note: *Ligands with 25% or more overall isolationyield in the LEAP testing were considered as positive

What is claimed is:
 1. A method for obtaining a composition or anisolate of exosomes or microvesicles comprising: providing a liquid thatincludes exosomes or microvesicles; providing a substrate having asurface, the surface having an array of exosome-binding ligands; whereineach ligand is in the form of an anionic group at pH 4-8; and whereinthe array of exosome-binding ligands enables exosomes or microvesiclesto bind to the substrate; contacting the liquid with the substrate inconditions enabling the binding of exosomes or microvesicles to theexosome-binding ligands; separating the substrate from the liquid;eluting exosomes or microvesicles from the exosome-binding ligands torelease the exosomes or microvesicles from the substrate after thesubstrate is separated from the liquid; separating the released exosomesor microvesicles from the substrate; thereby obtaining a composition orisolate of exosomes or microvesicles.
 2. The method of claim 1, whereinthe array is formed from one or more polymers having the exosome-bindingligands.
 3. The method of claim 2, wherein the one or more polymersinclude a monomer species having at least one exosome-binding ligand. 4.The method of claim 3, wherein at least 25% of the monomers of the oneor more polymers includes an exosome-binding ligand.
 5. The method ofclaim 3, wherein all monomers of the one or more polymers have anexosome-binding ligand.
 6. The method of claim 1, wherein at least 60%of ligands of the array are spaced no more than 10 angstrom apart fromanother ligand of the array.
 7. The method of claim 6, wherein at least60% of ligands of the array are spaced apart by more than 2 angstromsfrom another ligand of the array.
 8. The method of claim 7, wherein twoor more monomer species include an exosome-binding ligand.
 9. The methodof claim 1, wherein part of the substrate surface includes the array.10. The method of claim 2, wherein the polymer is a polysaccharide orpeptide.
 11. The method of claim 2, wherein the polymer is a syntheticpolymer.
 12. The method of claim 2, wherein the one or more polymersform the substrate.
 13. The method of claim 2, wherein the one or morepolymers are coupled to the substrate.
 14. The method of claim 2,wherein the one or more polymers are soluble in the liquid.
 15. Anapparatus for obtaining a composition or an isolate of exosomes ormicrovesicles by the method of claim 1 wherein the apparatus includes asubstrate as defined in claim
 1. 16. The method of claim 1, wherein theanionic group is selected from the group comprising: sulfates,selenates, phosphates, phosphonates, phosphinates, sulfated alcohols,amines, amides, sulfonamides, carboxylate groups, alcohols, ethers,anhydrides, imides, acylsulfonamides and combinations thereof.
 17. Themethod of claim 1, wherein the anionic group is selected from the groupcomprising: CO₂H, CH₂OH, CH₂OSO₃H, B(OH)₂, CH₂OP(O)(OH)₂, ionized formsthereof, and combinations thereof.
 18. The method of claim 7, wherein atleast 60% of ligands of the array are spaced apart by 3.5 to 6angstroms.
 19. The method of claim 7, wherein at least 60% of ligands ofthe array are spaced apart by 4 to 6 angstroms.
 20. The method of claim7, wherein at least 80% of ligands of the array have the definedspacing.
 21. The method of claim 20, wherein at least 90% of ligands ofthe array have the defined spacing.
 22. The method of claim 1, whereinthe array is planar and includes a region of exosome-binding ligandshaving a density of 1 to 10 ligands per nm².
 23. The method of claim 22,wherein the array includes a region of exosome-binding ligands having adensity of 5 ligands per nm².
 24. A composition or an isolate ofexosomes or microvesicles obtainable or obtained by the method of claim1.